Evolutionary conservation of KLF transcription factors and functional conservation of human γ-globin gene regulation in chicken

Genomics 84 (2004) 311 – 319 www.elsevier.com/locate/ygeno

Evolutionary conservation of KLF transcription factors and functional $ conservation of human g-globin gene regulation in chicken Priyadarshi Basu, a Thanh Giang Sargent, a Latasha C. Redmond, a Jeremy C. Aisenberg, a Evan P. Kransdorf, b Shou Zhen Wang, b Gordon D. Ginder, a,b,c and Joyce A. Lloyd a,b,* a

Department of Human Genetics, Virginia Commonwealth University, P.O. Box 980033, 1101 E. Marshall Street, Richmond, VA 23298-0033, USA b Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA c Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA Received 25 November 2003; accepted 18 February 2004 Available online 21 April 2004

Abstract The Kru¨ppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins with homology to Drosophila Kru¨ppel. KLFs can bind to CACCC elements, which are important in controlling developmental programs. The CACCC promoter element is critical for the developmental regulation of the human g-globin gene. In the present study, chicken homologues of the human KLF2, 3, 4, 5, 9, 11, 12, 13, and 15 genes were identified. Phylogenetic analysis confirms that these genes are more closely related to their human homologues than they are to other chicken KLFs. This work also represents the first systematic study of the expression patterns of KLFs during erythroid development. In addition, transient transfections of human globin constructs into 5-day (primitive) chicken red blood cells show that human g-globin expression is regulated via its CACCC promoter element. This indicates that a CACCC-binding factor(s) important for g-globin expression functions in 5-day chicken red cells. D 2004 Elsevier Inc. All rights reserved. Keywords: KLF; Chicken; Globin; Gene regulation; CACCC; Transcription factor

Kru¨ppel is a transcription factor in Drosophila melanogaster that plays a critical role in embryogenesis [1]. Among the large number of mammalian genes that exhibit sequence homology to the DNA-binding domain of Kru¨ppel, the Kru¨ppel-like factors (KLFs) are a particularly closely related family. The KLFs have three Cys2His2 (C2/H2) zincfinger domains and share conserved residues within and between these zinc fingers. There are 16 KLFs that have been identified in human, and they are related to the SP1-like family of transcription factors, with one overlap in nomenclature (SP6/KLF14) [2– 7]. Several KLFs are expressed in erythroid cells and are implicated in the expression of the globin genes. Erythroid Kru¨ppel-like factor (EKLF or KLF1) is a positive regulator of the h-globin gene and binds to the h-globin CACCC promoter element [8]. Other KLFs that are expressed in erythroid cells include KLF3/BKLF and KLF8/BKLF3, $

Supplementary data for this article may be found on ScienceDirect. * Corresponding author. Fax: (804) 828-3760. E-mail address: [email protected] (J.A. Lloyd).

0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.02.013

which bind to globin CACCC elements and repress transcription in transient transfection assays [9 – 11]. Also, KLF2/LKLF can activate the human h-globin gene promoter in transiently transfected mouse fibroblasts [12]. Chen and coauthors performed cDNA microarray analysis to establish the transcriptional program regulated by KLF4/GKLF in a colon cancer cell line (RKO) stably transfected with an inducible KLF4 gene. Interestingly, ~- and Ag-globin gene expression was up-regulated upon KLF4 induction [13]. Two Kru¨ppel-like factors (KLF11/FKLF1 and KLF13/ FKLF2) increase transcription of e-, g-, and h-globin promoter – reporter gene fusion constructs in transient transfection assays, and they require the CACCC element for this activity [14,15]. Recently, four KLF family members have been cloned from adult hematopoietic tissue in zebrafish. These include homologues of the mammalian KLF2, KLF4, and KLF12/AP2-rep [16]. Animal models employing ectopic expression or knockouts of the KLF genes have yielded important insights into their functional roles in development and differentiation. The neptune KLF is expressed in sites of primitive erythropoiesis

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in Xenopus, and it enhances globin induction in animal cap explants in conjunction with XGATA-1 [17]. Mice lacking Klf1 develop fatal anemia during fetal liver erythropoiesis, that is, Klf1 is not required for primitive erythropoiesis in the yolk sac but is required later in development [18,19]. In Klf1 / embryos containing the human g- and h-globin genes, the h-globin gene is not switched on as is normal in the fetal liver [20,21]. Klf2 / mutant mice die as embryos due to severe hemorrhaging caused by defective blood vessel morphology [22,23]. They also exhibit growth retardation and craniofacial abnormalities [23]. Wani and co-workers generated chimeric mice with Klf2 / ES cells and found that Klf2 is important for normal lung development [24]. Klf4 / mice die shortly after birth due to loss of skin barrier function, as measured by the penetration of external dyes and rapid loss of body fluids [25]. In another study, Klf4 has been shown to be essential for the terminal differentiation of goblet cells in the colon [26]. Klf5 / (Iklf / ) mice die before embryonic day 8.5 and Klf5 +/ mice show diminished levels of arterial wall thickening, angiogenesis, cardiac hypertrophy, and interstitial fibrosis in response to external stress [27]. Klf9 / (Bteb1 / ) mice mature normally and are fertile. These mice can survive for at least 2 years without evident pathological defects, after which they show deficits in motor coordination and in learning and memory tasks [28]. As it has not been tested directly, it is possible that Klf2, 4, 5, or 9 may have an effect on globin gene regulation in mice. Our laboratory has demonstrated that the CACCC and TATA elements in the human g-globin promoter are critical

for g-globin gene expression and inhibition of h-globin gene expression in transgenic mouse embryos. The ratio of h- to g-globin mRNA is higher in embryos containing constructs with mutations in the CACCC and TATA elements, both as a result of reduced g-globin mRNA and increased h-globin mRNA [29]. A stage-specific effect of the CACCC mutation in fetal cells was observed. It is now our objective to identify the factor(s) in the embryo that interacts directly with and regulates the g-globin gene through its CACCC promoter element. While KLF1 does not regulate the q- and g-globin genes, a factor with sequence similarity to KLF1 is likely to be involved. The first goal of the present work was to identify chicken KLFs and to determine the extent of conservation of KLFs between chicken and human. Eleven chicken KLF genes have been identified from the BBSRC chicken EST database, based on sequence homology to the human KLFs. The human and chicken KLF proteins are sufficiently similar to be potentially functionally equivalent. The mRNAs for some of these genes exhibit stage-specific erythroid expression. In the past, chicken h-like globin genes were shown to be developmentally regulated in mouse erythroid cells and in transgenic mice [30,31]. This work demonstrates the functional conservation of the regulation of the human g-globin gene in the chicken system. It was also shown that the factor(s) necessary for regulation of the human g-globin gene through its CACCC promoter element is functionally conserved in embryonic chicken RBCs.

Table 1 Sequence comparisons of chicken and human KLFs Chicken KLF

% homology with human in zinc-finger domain (S, similarity; I, identity) Peptide level

cKLF2

98.8% 97.6% 98.8% 97.6%

S I S I

100% 100% 100% 97.4% 98.8% 98.8% 100% 100% 92.3% 88.5% 100% 100% 98.4% 95.2% 97.6% 96.5% 88.2% 77.6%

S I S I S I S I S I S I S I S I S I

cKLF3

cKLF4a cKLF4b cKLF5 cKLF9 cKLF11 cKLF12 cKLF13 cKLF15a cKLF15b

Tissue sources of chicken ESTs from the BBSRC database

Nucleotide level

% homology with human over entire chicken sequence (length in nucleotides)

87.0%

56.7% (992)

83.9%

76.9% (968)

91.9%

90.0% (780)

Stage 20 whole embryo; Stage 36 head, limbs; adult small intestine, muscle, heart, brain, ovary Stage 10 whole embryo; Stage 20 whole embryo; Stage 22 limbs; Stage 36 limbs, trunk; adult kidney, liver Chondrocytes

87.5%

69.9% (861)

Stage 20 whole embryo; Stage 36 limbs

85.9%

65.9% (1313)

Stage 22 limbs; Stage 36 limbs; adult kidney

81.7%

60.9% (918)

Adult cerebrum, small intestine, heart, kidney

84.1%

56.9% (618)

Stage 22 head; Stage 36 trunks; adult ovary

88.2%

84.0% (1205)

Stage 36 head; adult ovary

84.1%

66.5% (453)

Stage 36 trunk; adult small intestine

81.6%

77.3% (608)

Adult ovary

74.9%

56.6% (682)

Adult liver, pancreas

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Results Identification of chicken KLFs Chicken homologues of the human KLF2, 3, 4, 5, 9, 11, 12, 13, and 15 genes were identified in the BBSRC chicken EST database, which contains approximately 340,000 chicken ESTs (Table 1). The identified ESTs were aligned to build contig/consensus sequences of the individual KLFs using the multiple sequence alignment program PRETTY. They were designated by the same KLF numbers as their human counterparts. Two chicken homologues of the human KLF4 (named chicken KLF4a and b) and KLF15 (named chicken KLF15a and b) genes were detected. The alignment of independent chicken KLF12 ESTs suggested that there are two isoforms of chicken KLF12, termed variants 1 and 2, with one variant missing 63 internal bases presumably due to alternative splicing, but with invariant zinc-finger domains.

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The sources of the ESTs, as listed in Table 1, suggest that the majority of the KLFs have a broad tissue distribution. Homologues for KLF1, 6, 7, 8, 10, 14, and 16 were not identified in the three existing chicken EST databases. It is possible that these genes are expressed at low levels during development, and therefore the ESTs have not yet been sequenced, or that these genes are not present in the chicken. To investigate further whether there is a chicken KLF1 gene, chicken genomic sequence in the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml) was interrogated with the human KLF1 mRNA sequence. Two genomic sequences were identified that could be potential candidates for chicken KLF1 (gnl/ti/260487286 tek99e12.g1 and gnl/ti/260288178 tdo98h10.g1). These sequences are more similar to human KLF1 than they are to any other KLF. However, there is no evidence from either the BBSRC EST database or our own RT-PCR data that either gene encodes an erythroid-specific mRNA (unpublished observation), as

Fig. 1. Alignment of the peptide sequences of the C2/H2 zinc-finger domains of the human, mouse, and chicken KLFs. The lines at the top indicate the three zinc fingers in this region. Dashes represent identical amino acids and asterisks indicate chicken amino acid sequence that is not available. The shaded amino acids are the invariant zinc-chelating residues, and the ‘‘^’’ indicates those residues that are believed to contact DNA. Similar and/or identical amino acids are represented by the same color. The chicken sequences were identified from the BBSRC database, and the human and mouse sequences were obtained from the NCBI database. Two distinct groups of ESTs similar to human KLF4 and KLF15 were identified and named ‘‘a’’ and ‘‘b’’, where the ‘‘a’’ gene is more similar to the human.

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do the human and mouse KLF1 genes. The best mechanism to resolve this issue will be the completion of the chicken genome project. Evolutionary conservation of the KLF genes across species The sequence alignments show that the KLFs are highly conserved across species (Table 1, Fig. 1). The amino acid sequences of the zinc fingers of each of the chicken KLFs are 88– 100% similar to the human KLFs. The similarity is striking in the zinc-finger domains of chicken KLF4a, KLF4b, KLF9, and KLF12, which have 100% peptide similarity with their human counterparts (Table 1). On average, the KLFs are 70% homologous between chicken and human over the entire available chicken EST sequence, which ranges from 453 to 1313 nucleotides. The most similar are chicken KLF4a and KLF12, which are 90 and 84% homologous to the human genes over 780 and 1205 nucleotides, respectively (Table 1). Fig. 1 shows the peptide alignment of the chicken KLF zinc fingers with their human and mouse homologues. Interestingly, the zinc-finger domains of KLF2, KLF5, and KLF12 are more similar between chicken and human than they are between mouse and human. The zinc-chelating C and H residues and the residues that come in contact with DNA [16] are conserved between all homologues for all KLFs (Fig. 1).The PXDLS domain [35], a CtBP binding motif present in human KLF12, is 100% conserved in chicken (QTEPVDLSINKA). This domain is also present in KLF2, but its presence in chicken could not be confirmed due to a lack of sufficient 5V sequence. The nuclear localization signal 1 domain [36 – 38], present in human KLF2 and KLF4 (KPKRGRRSWPRKR) immediately 5V of the zinc-finger domain, is also conserved in chicken. Unfortunately, there is not sufficient 5V sequence available for chicken KLF11 and 13 to determine if the mSin3A-interacting domain [39] is conserved with their human homologues. Phylogenetic analysis of the KLF zinc fingers revealed that the chicken KLFs cluster with their human counterparts rather than with other chicken KLFs (Fig. 2). The above evidence suggests that the functions of the chicken and human KLFs may be conserved as well. Expression of chicken KLF mRNAs during development Primers were designed for the chicken KLFs to determine their expression patterns through erythroid development by semiquantitative RT-PCR of RNA prepared from chicken blood cells. The time points chosen were 5, 7, and 14 days. Between 3 and 7 days the chicken expresses the embryonic U- and q-globin genes, and from day 8 the adult hH- and hAglobin genes are expressed. So the aforementioned time points coincide with primitive (5 days), transitional (7 days), and definitive (14 days) erythropoiesis. The chicken KLF2, 3, 4b, 9, 11, 12, and 13 mRNAs are expressed in blood cells (Fig. 3). All PCR products were sequenced to confirm

Fig. 2. Phylogenetic relationships of the zinc-finger domains of the human and chicken KLF proteins. The zinc-finger regions of the contig/consensus cDNAs were translated to peptide sequences. A neighbor-joining phylogram was constructed using the peptide sequences to demonstrate how the zinc-finger region is conserved between human and chicken proteins. The chicken proteins are designated as cKLFs and the human proteins as hKLFs. All of the human KLFs were included in this analysis, even if no chicken homologue of the gene has been identified.

correct amplification. For comparison, we also studied the expression patterns of these same KLFs in the developing brain (see supplementary figure). The expression of both isoforms of chicken KLF12 mRNA is significantly higher in primitive (5 and 7 days) than in definitive erythropoiesis (14 days). In fact, KLF12 mRNA is not detectable in 9-day blood

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Fig. 3. Semiquantitative RT-PCR expression data for chicken KLFs in chicken blood cells at various developmental stages. The time points are 5 (primitive), 7 (transition), and 14 days (definitive). Chicken GAPDH mRNA was used as an internal standard. The 5-day KLF/GAPDH ratio was set at 1 and the other time points were calculated relative to 5 days, to control for differences in the efficiency of the individual primer sets for the same mRNA. **Statistically significant difference of a 14-day value from the other two time points, using a rank sum test ( p < 0.05).

(unpublished observation). This developmental pattern of KLF12 mRNA expression may be erythroid specific, because the amount of KLF12 mRNA does not decrease during brain development. Expression of chicken KLF3 and KLF9 mRNA in blood is significantly higher at 14 days than at 5 or 7 days ( p < 0.05). Interestingly, KLF3, KLF9, and KLF11 mRNA is enriched between three- and fivefold in the blood compared to the brain. Expression of the other chicken KLFs does not vary significantly during erythroid or brain development, except that KLF13 mRNA increases more than fivefold between 5 and 14 days in the brain. Chicken KLF4a, 5, 15a, and 15b mRNA was not detected in blood. PCR primers for these genes were used with genomic DNA as template, the PCR products were sequenced, and it was confirmed that the designed primers were functional (unpublished observation). Therefore, if these four genes are expressed at all during erythropoiesis, their mRNA is present in very limited amounts. Chicken factors can bind to the human c-globin CACCC promoter element and regulate the gene via its CACCC box Electrophoretic mobility shift assay (EMSA) was performed to determine whether nuclear proteins from 5-day, 9-day, and adult chicken blood cells bind to the human g-

Fig. 4. EMSA with nuclear extracts from chicken blood cells. The assays represent nuclear extracts from 5-day (lanes 1 – 3), 9-day (lanes 4 – 6), or adult (lanes 7 – 9) chicken blood cells. The assays in lanes 1, 2, 4, 5, 7, and 8 are performed with a radiolabeled probe containing the wild-type human g-globin CACCC element. The sequence-specific complex in lanes 2, 5, and 8 is indicated by the arrow. This complex is competed away with 100 cold wild-type oligonucleotide used as a competitor (lanes 1, 4, and 7). The complex is not formed with a labeled probe with five base-substitution mutations in the CACCC element (lanes 3, 6, and 9).

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globin CACCC element. The results in Fig. 4 show that a sequence-specific complex binds to the CACCC probe (lanes 2, 5, and 8) at all three time points and is competed away with an excess of unlabeled oligonucleotide (lanes 1, 4, and 7). This complex is not formed with a probe with five basesubstitution mutations in the CACCC element (lanes 3, 6, and 9), confirming that the factor(s) binds to the CACCC box with sequence specificity. The functional effect of the CACCC element in globin expression was then investigated in a primary erythroid cell system. For the gene expression analysis, an HS2gh DNA construct or a similar construct containing a mutation in the g-globin CACCC element (HS2gcaccc*h) was transiently transfected into 5-day primitive chicken blood cells. These constructs contain the HS2 enhancer element from the h-globin locus control region and the intact human g- and h-globin genes. The HS2gcaccc*h construct has the same substitution mutation as in the mutant oligonucleotide probe used in EMSA. g-Globin gene expression is 11 times higher than h-globin expression in cells transfected with the HS2gh construct (Fig. 5), indicating that both human g- and h-globin genes are correctly regulated in these primitive chicken blood cells. When the HS2gcaccc*h construct is transfected, g-globin expression is 20-fold lower than with HS2gh ( p < 0.05), indicating that the CACCC element is essential for human g-globin expression in the chicken system. h-Globin expression is 3-fold higher in cells with the HS2gcaccc*h construct than with the HS2gh construct ( p < 0.05), indicating that the gand h-globin promoters actually compete for expression in this system. The overall g/h-globin mRNA ratio is only 0.17 in cells with HS2gcaccc*h, over 50-fold less than in cells transfected with HS2gh. This indicates that the protein(s) that binds to the g-globin CACCC element and is necessary for both g-globin expression and hglobin suppression is present in 5-day chicken blood cells.

Fig. 5. Chicken 5-day blood cells contain a factor(s) that regulates the human g-globin gene through the CACCC promoter element. A human HS2gh construct or a similar construct with a CACCC mutation in the gglobin gene promoter (HS2gcaccc*h) was transiently transfected into 5-day chicken blood cells. Real-time PCR was used to quantitate the amounts of human g- and h-globin mRNA in the transfected cells as described under Materials and methods. The amount of globin mRNA is expressed as a percentage of the mRNA from a cotransfected HLA-A2 gene.

Discussion Previous studies in our laboratory demonstrated that the CACCC and TATA elements in the g-globin promoter are critical for g-globin gene expression and inhibition of hglobin gene expression in transgenic mouse embryos. The objective of the current study is to determine the extent of structural conservation between the chicken and the human KLFs and also to determine if the function of the human gglobin CACCC element is conserved in chicken. In addition, this is the first systematic study of the expression patterns of the KLFs during erythroid development. Many KLFs have erythroid or embryonic expression patterns, as well as CACCC binding activity [2,3,5,7]. We expect that a factor(s) with sequence similarity to KLF1 may be involved in g-globin gene regulation. This factor is probably not KLF1 itself, since KLF1 is a positive regulator of the h-globin gene only and does not appear to have a direct effect on g-globin gene expression [19,21]. Chicken homologues of the human KLF2, 3, 4, 5, 9, 11, 12, and 13 genes were identified in the BBSRC chicken EST database. Phylogenetic and comparative sequence analyses of these proteins show that they are conserved between chicken, mouse, and human and therefore may have similar functions. The similarity is most striking in the zinc-finger domain, with four of the chicken KLFs having 100% peptide similarity with their human homologues. This result is similar to that of a phylogenetic comparison of the zebrafish, chicken, and human melanocortin receptor (MCR) genes [40]. The five MCR genes also cluster with their homologues, rather than with other related genes of the same species. In EMSA with nuclear extracts from chicken 5-day, 9-day, and adult blood cells, a sequence-specific complex binds to the human g-globin CACCC element. In transient transfection assays, human g-globin expression and suppression of h-globin transcription are observed in chicken 5-day blood cells, and this competition requires the g-globin CACCC element. This is complementary to the observation that the chicken globin genes are expressed at the correct developmental time points in transgenic mice [31]. So, although the genomic organization of the avian and mammalian h-globin loci is not very similar, it appears that control of gene regulation is well conserved. Correct regulation of the human globin genes in the transgenic mouse system also requires the g-globin CACCC element [29]. These observations suggest that the factor(s) that binds to and regulates the gglobin CACCC element is conserved in chicken, mouse, and human. It is noteworthy that there is a conserved lysine or leucine residue that contacts DNA in the third zinc finger of the KLFs (residue 76 in Fig. 1). It has been postulated that KLFs that have a leucine preferably recognize CACCC sequences [2,4]. Among the chicken KLFs, chicken KLF2, 3, 4a, 4b, 5, and 12 have a leucine, as do their human and mouse homologues, while chicken KLF9, 11, 13, 15a, and 15b and their homologues have a lysine in that position. The

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residue in this position is always conserved in mouse, human, and chicken, suggesting that the KLF homologues may have similar DNA-binding characteristics. It is significant that for three of the KLFs (KLF2, KLF5, and KLF12), the zinc-finger domain is more similar between chicken and human than between mouse and human. This helps to validate our theory that the chicken system can be used to study the roles of the KLF genes. Another interesting finding is that both chicken KLF12 transcripts are expressed only at the early time points (5 and 7 days) and not at 9 or 14 days, suggesting that they may function specifically in primitive erythropoiesis. This train of thought is also supported by the fact that both chicken KLF12 variants are expressed at steady levels throughout brain development. An alternative splicing event gives rise to the two transcripts of the chicken KLF12 gene. In humans, there are also two isoforms of the KLF12 mRNA that are alternatively spliced at an analogous position [41]. The zebrafish KLF4 and KLF12 genes are expressed during primitive erythropoiesis [16]. As discussed above, chicken KLF12 is expressed during primitive but not definitive erythropoiesis. The neptune KLF is closely related to KLF2 and KLF4 and acts as a positive regulator of primitive erythropoiesis in Xenopus. Interestingly, it also enhances globin induction in conjunction with Xenopus GATA-1 [17]. Taken together, these studies suggest that KLF2, 4, and 12 are strong candidates for positive regulation of the human embryonic (q) and/or fetal (g) globin genes. There are no ESTs for chicken homologues of the human KLF1, 6, 7, 8, 10, 14, and 16 genes in the three available chicken databases. It is possible that upon further expansion and refinement of the existing EST databases and upon availability of the chicken genome sequence, it will be possible to identify these KLFs. It is also possible that there are no chicken homologues for these KLFs. Although two genomic sequences that could be potential KLF1 candidates were identified, unlike KLF1 in human and mouse, these genes are not expressed in an erythroid-specific manner. The completion of the chicken genome sequencing project in the future should help resolve whether there is a chicken KLF1 gene. Some chicken KLFs may perform multiple functions that require additional KLFs in human, especially since KLFs appear to have evolved through multiple gene duplication events [7]. Similarly, some human KLFs may perform the functions of two or more chicken KLFs. A significant finding in our study was the identification of two chicken homologues for the KLF4 and KLF15 genes. KLF4 is expressed in multiple tissues during development and differentiation in the mouse [25,26]. The fact that chicken KLF4a is not expressed in chicken blood while chicken KLF4b is abundantly produced throughout erythroid development points to a division of function for these two genes. A major drawback in understanding and identifying the regulatory factors in globin regulation has been the lack of a suitable model system for the isolation of large numbers of

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nucleated primitive erythroid cells. The chicken is an excellent system for study of globin regulation by virtue of the presence of nucleated red blood cells and the relative ease with which primitive and definitive red cells can be isolated. This study shows that the mechanism of g-globin gene regulation is functionally conserved in human and the chicken. This validates the use of the chicken system for identifying human g-globin CACCC binding factors.

Materials and methods Data mining and bioinformatics A BBSRC chicken EST project database (http://www. chicken.umist.ac.uk/index.html) search was used to identify members of the chicken KLF family homologous to the human KLFs (from the NCBI database). Two other databases, the University of Delaware EST database (http:// www.chickenest.udel.edu) and the Bursal EST database (http://swallow.gsf.de/DT40/dt40Est.html), were also searched, but did not yield additional information. Several chicken homologues of each of the KLF genes were identified and aligned to build contig/consensus sequences using PRETTY, in the GCG Wisconsin package. The zinc-finger regions of these cDNAs were translated to peptide sequences using TRANSLATE. Alignments of the zinc-finger region of the human, mouse, and chicken homologues were done using PILEUP, and a neighbor-joining phylogram was drawn using GROWTREE from the same package. The ID numbers of chicken ESTs from the BBSRC database used to build the contig/consensus sequences are KLF2, ChEST618p12, ChEST991d8, ChEST53o1, ChEST62g24, ChEST383n2, ChEST512i9, ChEST879d8, ChEST679m9, ChEST596p22, ChEST596p8, ChEST221n21; KLF3, ChEST646h3, ChEST419i12, ChEST549e9, ChEST432d9, ChEST423c13, ChEST872j13, ChEST915e11, ChEST369j5, ChEST 248m18, ChEST820l24; KLF4a, ChEST217h11; KLF4b, ChEST972g23, ChEST913e4; KLF5, ChEST429a18, ChE ST377m3, ChEST962a13, ChEST806h6, ChEST804h3, ChEST816g2; KLF9, ChEST544o17, ChEST624d4, ChEST511j15, ChEST797b8; KLF11, ChEST866m2, ChEST296m22, ChEST698o10; KLF12, ChEST396c13, ChEST198l24, ChEST817c2; KLF13, ChEST530j10, ChEST150e16; KLF15a, ChEST276o17, ChEST694k12; and KLF15b, ChEST736o15, ChEST942p10. The contig/ consensus sequences have been submitted as supplementary material. RNA isolation and semiquantitative RT-PCR Semiquantitative RT-PCR of the chicken KLF mRNAs was performed with total RNA from 5-, 7-, and 14-day chicken blood cells. Chicken blood cells were harvested and RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol with one modification; the

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extraction was repeated two additional times. The isolated RNA was treated with 2 units of RQ1 DNase (Promega) per microgram of RNA for 30 min at 37jC. Brain RNA was isolated using a standard protocol and treated with DNase prior to PCR. PCR controls were included to confirm that no genomic DNA was amplified. Primers for PCR were designed using PRIME, a primer-generation software in the GCG Wisconsin package. Primer sequences are available upon request. Each data point in the RT-PCR graphs represents an average of at least two different reactions carried out with two separate sets of primers for a total of at least four reactions, except for chicken KLF12, for which only one primer set was used. All PCR products were sequenced to confirm amplification of the correct mRNA. Chicken GAPDH mRNA was used as an internal standard. The 5day KLF/GAPDH ratio was set at 1, and the other time points were calculated relative to 5 days, to control for differences in the efficiency of the individual primer sets for the same mRNA. Statistical analysis of the fold change of KLF expression was done using a rank sum test, and the reported differences were significant at p < 0.05. Nuclear extract preparation and binding assays EMSA was performed with nuclear extracts from chicken blood. Blood cells were collected using standard Histopaque (Sigma Diagnostics) separation. Nuclear extracts were made using the Dignam method [32] with one additional step. The cells were washed three times with buffer A to remove excess hemoglobin before they were lysed by Dounce homogenization. For EMSA, an oligonucleotide containing the normal sequence of the CACCC element in the g-globin promoter (5V-CCCTGGCTAAACTCCACCCATGGGTTGGCC-3V) and its complementary sequence were annealed to make the wild-type probe. The corresponding CACCC mutant oligonucleotide (5V-CCCTGGCTAAACTAGTACTATGGGTTGGCC-3V) and its complementary sequence were also annealed. The probes were end-labeled with [32P]ATP using T4 polynucleotide kinase (New England Biolabs). The binding buffer consisted of 20 mM Tris, pH 7.5, 60 mM KCl, 0.5 mM EDTA, 2 mM DTT, 5 mM h-mercaptoethanol, and 5% glycerol. The binding reactions were performed on ice using poly(dI/dC) as the nonspecific competitor. Unlabeled wildtype oligonucleotide in 100-fold excess to the probe was used as a sequence-specific competitor in some reactions. The samples were run on 5% nondenaturing polyacrylamide gels at 12 V/cm and 4jC. Transient transfection assays and real-time PCR For the gene expression analyses, an HS2gh DNA construct and a similar construct containing a substitution mutation in the g-globin CACCC element (the same mutation as in the oligonucleotide for the EMSA experiments; HS2gcaccc*h) were transiently transfected into 5-day primitive chicken blood cells. The wild-type construct is the same

as used previously [33], except that an additional 750-bp HindIII fragment 3V of the g-globin gene is included. The mutant plasmid was generated by site-directed mutagenesis of the wild-type construct using a Clontech kit. For each assay, 108 cells were subjected to osmotic transfection [34]. Real-time PCR was used to calculate the amount of g- and hglobin mRNA from the transfected genes. The human HLAA2 gene driven by a CMV promoter was cotransfected with the globin constructs and was used as an internal standard for the PCRs and as a control for transfection efficiency. The primer and probe sequences used are h-globin forward primer, 5V-GCAAGGTGAACGTGGATGAAGT-3V; h-globin reverse primer, 5V-TAACAGCATCAGGAGTGGACAGA-3V; h-globin TaqMan probe, 5V-CAGGCTGCTGGTGGTCTACCCTTGGACCC-3V; g-globin forward primer, 5V-GTGGAAGATGCTGGAGGAGAAA-3V; g-globin reverse primer, 5V-TGCCATGTGCCTTGACTTTG-3V; g-globin TaqMan probe, 5V-AGGCTCCTGGTTGTCTACCCATGGACC-3V; HLA-A2 forward primer, 5V-ACCTGGAGAAGCGGAAGGA-3V; HLA-A2 reverse primer, 5V-CAGAGACAGCGTGGTGAGTCA-3V;and HLA-A2 TaqMan probe, 5V-CGCACGGACGCCCCCAA-3V. The primers and probes for the g- and the h-globin mRNAs were calculated to have similar efficiencies. Each of the three probes spans an exon –exon junction to eliminate the possibility of signal from amplified genomic DNA. The amount of human globin mRNA is expressed as a percentage of HLAA2 mRNA. Each data series represents the mean of eight transfections using two different plasmid DNA preparations. Statistical analysis was done using a rank sum test, and the results were significant at p < 0.05.

Acknowledgments We are grateful to Pamela Morris for excellent technical assistance. We thank Andrew Chervenak for his work on the chicken KLF1 candidates. This work is supported by NIH Grant HL60080 to J.A.L.

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